Post on 02-Jan-2017
Chapter 8Enzymes: basic concepts and kinetics
Light production by enzyme catalysis: energy conversion
Outline:
• enzymes are powerful and specific catalysts
• free energy is a useful thermodynamic function for understanding enzyme catalyzed reactions
• enzymes accelerates reactions by facilitating the formation of the transition state
• the Michaelis-Menten model accounts for the kinetic properties of many enzymes
• enzymes can be inhibited by specific molecules
Reaction catalyzed by a proteolytic enzyme
Enzymes are specific catalysts (8.1)
The peptide bond is thermodynamically unfavorable but kinetically very stable (half-life 1000 years). Why?
Specificity of enzyme catalysis
Trypsin and thrombin are both proteases.
• Trypsin functions in the degradation of ingested proteins
• Thrombin acts only on one protein: fibrinogen. Thrombin converts fibrinogen into fibrin and fibrin polymeri-zes and forms a blood clot.
Thrombin
Trypsin
Enzymes are powerful catalysts (8.1)
HCO3- + H+
Reaction catalyzed by carbonic anhydrase
Why is a reaction that occurs spontaneously catalyzed by an enzyme?
Stimulates O2 release from Hb
Fysiologische rol koolzuur anhydrase
[Figuur 7.22]
pH < 7 pH ~ 7.4
Rate enhancement by enzymes
Carbonic anhydrase is one of the fastest enzymes known. One enzyme molecule can hydrate 106
molecules of CO2 per second.
Many enzymes require cofactors for activity
Apoenzyme + cofactor = holoenzymeCofactors that are (small) organic molecules are called coenzymes.
If coenzymes are tightly bound, they are called prosthetic groups.
If coenzymes are only bound during catalysis, they are called cosubstrates.
Metals like Zn, Ni are tightly bound.
cofactor
metalen coenzym
prosthetische
groep cosubstraat
De covalent gebonden cofactor, pyridoxalfosfaat, (coenzym) die een prosthetischegroep is van het enzym glycogeen fosforylase
Zink, een covalent gebonden cofactor aan hetenzym koolzuur anhydrase
Animatie
Enzymes can transform energy from one form into another
• Pomp heeft twee conformaties. • Een met de ion bindingsplaatsen open voor een kant
van het membraan.• De andere conformatie is open voor de andere kant. • Een gekoppelde vrije energie leverende reactie
verschuift het evenwicht tussen de twee conformaties.
Figuur 13-2
Enzymes can transform energy from one form into another
Structuren van de calcium pomp
Calciumbindingsplaatsen bereikbaar vanuit het cytoplasma [Fig 13-3]
Calciumbindingsplaatsen niet meer aanwezig en open naar de ander kant van het membraan [Fig 13-4]
Enzymes can transform energy from one form into another
The Ca2+ ATPase uses the energy of ATP hydrolysis to transport Ca2+
across the membrane, generating a Ca2+
gradient.
Figuur 13-5
Free energy is a useful thermodynamic function for understanding enzyme catalyzed reactions
(8.2)The free energy difference of the products minus that of the substrates of a reaction determine the direction of the reaction (ΔG).
]].[[]].[[ln0
BADCRTGG +Δ=Δ
The reaction: A + B C + D
Free energy is a useful thermodynamic function for understanding enzyme catalyzed reactions (8.2)
Een reactie kent twee thermodynamische eigenschappen
• Het vrije energieverschil tussen de producten en de reactanten bepaalt of een reactie spontaan kan verlopen (ΔG).
• De vrije energie die nodig om de activeringsenergie te overwinnen bepaalt hoe snel een reactie verloopt (ΔGǂ).
At pH 7 add '
]].[[]].[[ln0''
BADCRTGG +Δ=Δ
R = gas constant = 8.315 10-3 kJ. mol-1. K-1
T = temperature in K, 25 °C = 298 °Kln x = 2.303 log xTogether RTlnx = 5.71logx
At equilibrium]].[[]].[[ln0 0''
BADCRTGG +Δ==Δ
eqKRTG ''0 ln−=Δ
7150 ./log '' −Δ= GK eq 715010 ./''
]][[]][[ G
eqBADCK Δ−==
If Δ G0' = -5.71 kJ.mol-1, the equilibrium shifts a factor of 10 to the right!!! Hydrogen bonds have energies ranging from 4 to 20 kJ.mol-1
Calculate the free energy for the isomerization of DHAP to GAPAt equilibrium the ratio GAP/ DHAP is 0.0475 at 25 °C and pH 7, calculate the standard free energy.
• ΔG0' = -2.303 x R x T x log K'eq= -2.303 x 8.315 x 10-3 x 298
x log (0.0475) = 7.55 kJ mol-1
• The reaction is endergonic under the standard conditions.
• What will happen when the concentration DHAP is 2 x 10-4 M and GAP is 3 x 10-6 M?
• ΔG' = 7.55 + 2.303 x RTlog([GAP]/[DHAP])= 7.55 – 10.42= -2.87 kJ mol-1
• The reaction is exergonic.
The criterion of spontaneity is determined by the ΔG and not by the
ΔG0'.
• The free energy difference of the products minus that of the substrates of a reaction determine the direction of the reaction (ΔG)
Enzymes accelerate reactions by facilitating the formation of the transition state (8.3)
• The free energy of activation of a reaction determines the rate of a reaction (ΔG‡)
v = factor x [s] x e-ΔGǂ/RT
• The essence of catalysis is optimizing the structure for the interactions of the transition state.
• If the transition state is stabilized by 5.71 kJ.mol-1, the rate increases 10 fold.
Bindingsenergie wordt gebruikt om de DNA dubbelhelix te buigen waardoor water de fosfodiesterbinding kan bereiken en hydrolyseren
Figuur 9.38 en 9.42
The formation of an enzyme-substrate complex is the first step in enzymatic catalysis
The formation of an enzyme-substrate
complex (ES)
E + S ES E + Pk1
k-1 k-2
k2
The three dimensional structure of the catalytic subunit of protein A kinase. The inhibitor contains a pseudosubstrate sequence
Arg-Arg-X(Asn)-Ala(Ser,Thr)-Ile
Properties of the active sites of enzymes
• The active site is a three dimensional cleft or crevice
• The amino acid residues involved in binding the substrate(s) are called the catalytic groups
• The active site takes a relative small part of the total volume of an enzyme
• Substrates are bound to enzymes by multiple weak interactions
• The specificity of binding depends on the precisely defined arrangement of atoms in an active site
• Enzymes are flexible and the active site can be formed by the binding of the substrate (induced fit), extra free energy when bound water is liberated
The active site can be formed by amino acid residues from different parts of the polypeptide chain
Ribbon diagram (A) and a schematic representation of the primary structure of
lysozyme (B)
Hydrogen bonds between ribonuclease (enzyme) and the uridine component of its
substrate induce a high degree of specificity
Substrates are bound by multiple weak interactions:
• electrostatic interactions• hydrogen bonds• van der Waals forces• hydrophobic interactions
258081010 71511257150=== Δ− ./../'' G
eqK
Three hydrogen bonds ~ 3*-8.4 = - 25.11 kJ.mol-1. Change in equilibrium about 25000 times
-CH3group in thymine
Enzyme kinetics: the Michaelis-Menten model (8.4)
• The study of the rates of chemical reactions is called kinetics.
• The study of the rates of enzyme-catalyzed reactions is called enzyme kinetics.
• A kinetic description of enzyme activity (v) will help understand how enzyme functions.
Rate of a chemical reaction
S P rate (v) = - ΔS/Δ T = k x [S]; k = rate constantk
Rate of an enzyme catalyzed reaction
The formation of an enzyme-substrate complex is followed by product formation
E + S ES E + P
k1
k-1 k-2
k2
The rate the reaction is the rate of product formation
Determination of the rate as a function of the substrate concentration
The initial velocity (V0) is determined under steady state conditions
E + S ES E + Pk1
k-1 k-2
k2
Enzyme catalysis with formula
E + S ES E + Pk1 k2
k-1
v = k2[ES]
v = Vmax [S]/([S] + KM)
KM = (k2 + k-1)/k1
Vmax = k2 [ET]
Vmax = kcat [ET]
The significance of KM (KM = (k2 + k-1)/k1)
• KM values vary between 10-7 M en 10-1 M
• KM value is the substrate concentration with half of the binding sites occupied (half maximal velocity)
• The KM value is an indication of the substrate concentration in vivo
- kcat of an enzyme is the number of substrate molecules that is converted per second into product per enzyme molecule under saturating substrate concentrations
- kcat is also called the turnover number. Vmax = kcat[ET]
- kcat is a direct measure of the catalytic capacity of an enzyme under saturating substrate concentrations
- 1/kcat is time of a complete catalytic cycle.
The significance of kcat
Most biochemical reactions include multiple substrates
• Sequential reactions
Ternary complex
Most biochemical reactions include multiple substrates
• Double-displacement (Ping-Pong) reactions
Allosteric enzymes do not obey Michaelis-Menten kinetics
M-M kinetics Allosteric kinetics
Distinction between competitive, uncompetitve and noncompetitive inhibition (reversible inhibition)
Enzymes can be inhibited by specific molecules (8.5)
Competitive, uncompetitive and noncompetitive inhibition are kinetically
distinguishable
Competitive Uncompetitive Noncompetitive
Chapter 15 Metabolism: Basic Concepts and Design
Outline of chapter 15
• Metabolism is composed of many coupled andinterconnected reactions
• ATP is the universal currency of free energy in biological systems
• The oxidation of carbon fuels is an important source of cellular energy (redox reactions)
• Metabolic pathways contain many recurring motifs(the unifying themes of biochemistry)
Living organisms require a continual input of free energy for:
• the performance of mechanical work in muscles and other cellular movements
• active transport of molecules and ions
• the synthesis of macromolecules
The free energy is derived from:
• Sunlight: phototrophs are trapping sunlight in photosynthesis (conversion of energy-poor molecules like CO2 into energy-rich molecules like fatty acids and sugars).
• Oxidation of compounds (foodstuffs): chemotrophs oxidize (carbon) compounds. Foodstuffs are generated by phototrophs.
Metabolism is composed of many coupled and interconnected reactions (15.1)
Glucose metabolism in humans•Glucose is metabolized to pyruvate in 10 linked reactions.
•Under anaerobic conditions pyruvate is metabolized to lactate (2 ATP).
•Under aerobic conditions pyruvate oxidized to CO2 and H20 via acetyl CoAand the TCA cycle and respiratory chain (30 ATP).
An example of a metabolic pathway: glycolysis. The free energy of the overall process must be
negative.
All reactions are catalyzed by enzymes. The activity of the glycolysis is regulated.
Free energy of metabolites of glycolysis in red blood cells
(138) = concentration in μM
-
-
-
-
Metabolic pathways can be divided into:
• Catabolic reactions: catabolism: fuels (carbohydrates, fats) CO2 + H2O + useful energy
• Anabolic reactions: anabolism: useful energy + small molecules complex molecules
• Some pathways can be either anabolic or catabolic, depending on the energy conditions of the cell. They are referred to as amphibolic pathways
De citroenzuurcyclus als amfibole route
De citroenzuurcyclus wordt gebruikt om acetyl-groepen af tebreken (katabolisme), maar dient ook als bron voor biosynthese(anabolisme).De omzetting van pyruvaat naar oxaloacetaat is hiervoor vereist.
Een anaplerotische reactie
Een belangrijke reactie in de vorming van glucose uit amino-en ketozuren is de carboxylering van pyruvaat tot oxaloacetaat. Deze reactie is gekoppeld aan ATP hydrolyse en wordt gekatalyseerd door het biotine bevattende enzym pyruvaat carboxylase. De door pyruvaat carboxylasegekatalyseerde reactie verloopt in drie stappen:
1) HCO3- + ATP HOCO2-PO3
2- + ADP
2) Biotine-enzym + HOCO2-PO32- CO2-biotine-enzym + Pi
3) CO2-biotine-enzym + pyruvaat biotine-enzym + oxaloacetaat
Somreactie:Pyruvaat + HCO3
- + ATP oxaloacetaat + ADP + Pi + H+
• De overall reactie heeft een standaard vrije energie van 0.8 kJ. mol-1.
• De hydrolyse van ATP tot ADP en Pi heeft een standaard vrije energie van -31.4 kJ. mol-1.
• In de reactiecyclus wordt CO2 geactiveerd. De splitsing van CO2 van het CO2-biotine-enzym complex heeft een standaard vrije energie van -19.3 kJ. mol-1. Dit hoog energetisch intermediair wordt gebruikt om pyruvaat te carboxyleren.
• Welk belangrijk principe wordt door pyruvaat carboxylasegedemonstreerd?
A B ΔG0' = + 16.7 kJ mol -1
K'eq = [Beq]/[Aeq] = 10 - ΔG0'/5.71 = 1.19 x 10-3 = 1 / 841
At equilibrium 841 molecules of A and 1 molecule B or a protein in conformatie A or B!!
ATP hydrolysis drives metabolism or can perform work by shifting the equilibrium of coupled
reactions
Coupled with ATP hydrolysis (ΔG0' = -30.6 kcal mol -1)
A + ATP + H2O B + ADP + Pi + H+
(ΔG0' = 16.7 – 30.6 = -13.9 kJ mol -1)
K'eq = [Beq]/[Aeq] x ([ADP]eq [Pi]eq)/[ATP]eq
= 10 - ΔG0'/5.71 = 2.72 x 102 M-1
The ATP-generating system in the cells maintains the ATP]/[ADP][Pi] ratio around 500 M-1. With this ratio is the equilibrium between A and B is shifted further towards excess B.K'
eq x [ATP]cel/([ADP]cel [Pi]cel)= [Beq]/[Aeq][Beq]/[Aeq] = 2.72 x 102 x 500 = 1.36 x 105
Thus by coupling with ATP hydrolysis the ratio [A]/[B] shifts from
1360001
1841to
BA=
Why is ATP an energy-rich molecule?
• ATP + H2O ADP + Pi + H+ : ΔG0’ = - 30.6 kJ/mol
• ADP + H2O AMP + Pi + H+ : ΔG0’ = - 30.6 kJ/mol
• AMP + H2O adenosine + Pi + H+ : ΔG0’ = -14.3 kJ/mol
The structural basis of the high phosphoryl transfer potential of ATP
•Resonance stabilization•Electrostatic repulsion•Stabilization of phosphate by hydration
Free phosphate (4x) has more energetic favorable resonance structures compared with the terminal phosphate of ATP or ADP
3x 2x 2x
The amount of ATP is limited. ATP is continuously regenerated
The ATP-ADP cycle
• 100 g ATP in your body
• In rest 40 kg turnover in 24 hours. Turnover 3.6 min
• Running: 500 g / min. Turnover 0.2 min
The oxidation of carbon fuels is the only source of cellular energy for animals but not for microorganisms (15.3)
(H2 gas)
Free energy of oxidation of single carbon compounds
In aerobic organisms the electron acceptor in the oxidation of carbon and hydrogen is O2 and the oxidation products
are CO2 and H2O
Fats are more efficient fuel source than carbohydrate because carbon is more
reduced
Stages in the extraction of
cellular energy from foodstuffs,
mainly reducing equivalents
8 e- = 3 x NADH, 1 x FADH2
Extraction of free energy in the form of ATP from fuel molecules
• In catabolism, some ATP is generated (substrate level phosphorylation), but most of the free energy is temporary stored in the reducing equivalentsextracted from fuel molecules.
• The reducing equivalents are transferred to NAD+
and FAD. NADH and FADH2 are formed.
• Reducing equivalents are transferred to an electron transport chain, a respiratory chain.
• Free energy is stored in a proton gradient that drives the synthesis of ATP.
Oxidation can be coupled directly to ATP synthesis
Energy of oxidation is trapped as a high phosphoryl-transfer-potential compound and then used to form ATP
De overgang van een C-H naar eenC-OH binding produceert58.6 kJ . mol-1
Electron transport chains generates ion gradients across membranes providing an important form of
cellular energy that can be coupled to ATP synthesis
The total process is called oxidative phosphorylation
NADH, FADH2
High-energy electrons: redox potentials and free-energy changes
'' EnFG Δ−=Δ
'0
'0 EnFG Δ−=Δ
The relation between a redox potential change and the free energy change of a reactions is:
Redoxpotential
-
+
F = 96.49 kJ.V-1.mol-1
Redox potential (ΔE) and free energy (ΔG)
• A 1.14-volt potential difference between NADH and O2 drives electron transport through the respiratory chain. This electron transport is coupled to the formation of a proton gradient (ΔG0 = -2 x 96.49 x 1.14 = -220 kJ.mol-1)
• A strong reductant has a negative reduction potential, a strong oxidizing agent has a positieve reduction potential
Standard reduction potentials
of biological important reactions
The mitochondrial electron transport chain,bacterial respiratory chains function essential
similar
ATP synthesis from a proton gradient
Comparison between photosynthesis and
oxidative phosphorylation
Figuur 19.25
Licht wordt gebruikt om electronen naar een sterkere reductor over te brengen
Figuur 18.6 Figuur 19.23
Metabolic pathways contain many recurring motifs (15.3)
• Activated carriers
• Key reactions are reiterated
• Metabolic processes are regulated in only three principle ways
Activated carriers of electrons for fuel oxidation Structures of the
oxidized forms of nicotinamide-derived electron carriers
• R = H: NAD+
• R= PO3-: NADP+
• The nicotinamide ring of NAD+
accepts a hydrogen atom and two electrons, which is equivalent to a hydride ion, H-
NAD+
NADPH is the reductant in biosynthesis,NADH is used primarily for the generation
of ATP
Flavin adenine dinucleotide is an electron carrier
• FAD consists of flavin mononucleotide and an AMP unit
• The molecule accepts 2 electrons and 2 protons
The redox reaction of FAD
Redox reactions and the involved redox carriers (NAD(P)+)
• The redox reaction catalyzed by NAD+ dependent redox enzymes.
• NAD+ always functions as coenzyme (cosubstrate)
H- (hydride) transfer
Redox reactions and the involved redoxcarriers (FAD)
• The redox reaction catalyzed by FAD dependent redox enzymes
• FAD is always bound to the enzyme (prosthetic group)
H (hydrogen) transfer
Coenzyme A is the carrier for activated acyl groups
ΔG0' of hydrolysis is -30.6 kJ mol-1CoA
Carriers used in metabolism
The activated carriers are (kinetically) stable
Key reactions are reiterated throughout metabolism
The six fundamental reactions types are the basis of all reactions of metabolism
Oxidation-reduction reactions
Ligation reactions form bonds by using the free energy from ATP hydrolysis
Isomerization reactions
Group-transfer reactions
Hydrolytic reactions
The hydrolysis of a peptide bond
Lyases: enzymes that catalyze the addition or the removal of functional groups to/from
double bonds or the cleavage involving electron rearrangement
rearrangements
Metabolic processes are regulated in three different principle ways
• The amount of enzymes– rate of transcription
• The catalytic activity of the enzymes– reversible allosteric control– feed back inhibition– reversible covalent modification – hormones coordinate metabolic relations between different
tissues often via reversible covalent modification of key enzymes
• The accessibility of substrates– controlling the flux of substrates from one compartment to
another (e.g. cytosol to mitochondria).
Biosynthetic and degradative pathways are almost always distinct for energetic reasons
Regulation by the energy charge = ][][][][][ 2
1
AMPADPATPADPATP++
+
The evolution of metabolic pathways
• Why do activated carriers such as ATP, NADH, FADH2 and coenzyme A contain adenosine diphosphate units?
• Binding to a uracilunit in a niche of an RNA enzyme (ribozyme) in the RNA world
• In the protein world, the carrier function could be continued without any adaptation